Subcutaneous Glucose Electrode

ABSTRACT

A small diameter flexible electrode designed for subcutaneous in vivo amperometric monitoring of glucose is described. The electrode is designed to allow “one-point” in vivo calibration, i.e., to have zero output current at zero glucose concentration, even in the presence of other electroreactive species of serum or blood. The electrode is preferably three or four-layered, with the layers serially deposited within a recess upon the tip of a polyamide insulated gold wire. A first glucose concentration-to-current transducing layer is overcoated with an electrically insulating and glucose flux limiting layer (second layer) on which, optionally, an immobilized interference-eliminating horseradish peroxidase based film is deposited (third layer). An outer (fourth) layer is biocompatible.

This application is a Continuation application Ser. No. 11/109,379,filed Apr. 19, 2005, which is a Continuation application Ser. No.10/353,341, filed Jan. 28, 2003, now U.S. Pat. No. 6,881,551, which is aContinuation application of Ser. No. 09/997,808, filed Nov. 29, 2001,now U.S. Pat. No. 6,514,718, which is a Continuation application of Ser.No. 09/668,221, filed Sep. 22, 2000, now U.S. Pat. No. 6,239,161, whichis a Continuation of application Ser. No. 09/477,053, filed Jan. 3,2000, now U.S. Pat. No. 6,162,611, which is a Continuation ofapplication Ser. No. 09/356,102, filed Jul. 16, 1999, now U.S. Pat. No.6,121,009, which is a Continuation of application Ser. No. 08/767,110,filed Dec. 4, 1996, now U.S. Pat. No. 6,284,478, which is a continuationof application Ser. No. 08/299,526, filed Sep. 1, 1994, now U.S. Pat.No. 5,593,852, which is a continuation-in-part of application Ser. No.08/161,682, filed Dec. 2, 1993, now U.S. Pat. No. 5,356,786, which is acontinuation of application Ser. No. 07/664,054, filed Mar. 4, 1991, nowabandoned, which applications are incorporated herein by referenceherein.

This work was supported in part by the National Institutes of Health(DK42015). Accordingly, the U.S. Government may have rights in thisinvention.

FIELD OF THE INVENTION

The present invention relates to in vivo enzyme biosensors and morespecifically to miniature glucose sensors for subcutaneous measurementof glucose with one-point calibration.

BACKGROUND

In response to the need for frequent or continuous in vivo monitoring ofglucose in diabetics, particularly in brittle diabetes, a range ofpossible in vivo glucose electrodes have been studied. The desiredcharacteristics of these electrodes include safety, clinical

accuracy and reliability, feasibility of in vivo recalibration,stability for at least one hospital shift of eight hours, small size,ease of insertion and removal, and a sufficiently fast response to allowtimely intervention. The in vivo recalibration should be based uponwithdrawal of a single sample of body fluid, e.g., blood, and measuringits glucose concentration. This is termed “one point calibration”.

Keys to safety are absence of leachable components, biocompatibility,and limiting of the potentially hazardous foreign matter introduced intothe body to an amount that is inconsequential in a worst case failure.The clinical accuracy must be such that even when the readings are leastaccurate, the clinical decisions based on these be still correct.Feasibility of prompt confirmation of proper functioning of the sensorsand of periodic in vivo recalibration is of essence if a physician is toallow the treatment of a patient to depend on the readings of thesensor. This one-point calibration, relying on the signal at zeroglucose concentration being zero and measuring the blood glucoseconcentration at one point in time, along with the signal, is ofessence, but has heretofore been elusive. The sensitivity must besufficiently stable for the frequency of required in vivo recalibrationto not be excessive. The sensor must be small enough to be introducedand removed with minimal discomfort to the patient and for minimaltissue damage. It is preferred that the sensor be subcutaneous and thatit be inserted and removed by the patient or by staff in a physician'soffice. Finally, its response time must be fast enough so thatcorrective measures, when needed, can be timely.

In response to some of these needs, needle type and other subcutaneousamperometric sensors were considered. The majority of these utilizedplatinum-iridium, or platinum black to electrooxidize H₂O₂ generated bythe glucose oxidase (GOX) catalyzed reaction of glucose and oxygen. Inthese sensors, the GOX was usually in large excess and immobilized,often by crosslinking with albumin and glutaraldehyde. To excludeelectrooxidizable interferants, membranes of cellulose acetate andsulfonated polymers including Nafion™ were used. Particular attentionwas paid to the exclusion of the most common electrooxidizableinterferants: ascorbate, urate and acetaminophen. Also to cope with theinterferants, two-electrode differential measurements were used, oneelectrode being sensitive to glucose and electrooxidizable interferantsand the other only to interferants. One strategy for overcoming theproblem of interferants, applicable also to the present invention,involves their preoxidation. Another strategy involves shifting, throughchemical changes, the redox potential of the polymer in the sensinglayer to more reducing potentials. When the redox potential of thepolymer is in the region between about −0.15 V and +0.15 V versus thestandard calomel electrode (SCE), and the electrodes are poised in theirin vivo operation between about −0.10 and +0.25 V, the rate ofelectrooxidation of interferants such as ascorbate, urate, andacetaminophen is very slow relative to that of glucose through itsphysiological concentration range. Thus, also the currents fromelectrooxidation of interferants are small relative to those of glucose.

To make the electrodes more biocompatible, hydrophilic polyurethanes,poly(vinyl alcohol) and polyHEMA membranes have been used.

Several researchers tested GOX-based glucose sensors in vivo andobtained acceptable results in rats, rabbits, dogs, pigs, sheep andhumans. These studies validated the subcutaneous tissue as an acceptableglucose sensing site. Good correlation was observed betweenintravascular and subcutaneous glucose concentrations. They alsodemonstrated the need for in vivo sensor calibration. Another approachto in vivo glucose monitoring was based on coupling subcutaneousmicrodialysis with electrochemical detection. To control and adjust thelinear response range, electrodes have been made glucose-diffusionlimited, usually through glucose transport limiting membranes.

Diffusional mediators, through which the O₂ partial pressure dependenceof the signals is reduced, are leached from sensors. Such leachingintroduces an unwanted chemical into the body, and also leads to loss insensitivity, particularly in small sensors. In microsensors, in whichoutward diffusion of the mediator is radial, the decline in sensitivityis rapid. This problem has been overcome in “wired” enzyme electrodes,i.e., electrodes made by connecting enzymes to electrodes throughcrosslinked electron-conducting redox hydrogels (“wires”). Glucoseoxidase has been “wired” with polyelectrolytes having electron relaying[Os(bpy)₂Cl]^(+/2+) redox centers in their backbones. Hydrogels wereformed upon crosslinking the enzyme and its wire on electrodes. Theseelectrodes had high current densities and operated at a potential of0.3V vs. SCE. The electrooxidizable interferants are eliminated throughperoxidase-catalyzed preoxidation in a second, nonwired, hydrogenperoxide generating layer on the “wired” enzyme electrode.

SUMMARY OF THE INVENTION

A small (e.g., 0.29 mm), recessed, non-corroding metal (e.g., gold,platinum, palladium) or carbon wire electrode for subcutaneous in vivoglucose monitoring, approaching in its performance all of the abovelisted requirements, including in vivo one-point calibration, has beenproduced. The electrode was constructed by depositing active polymerlayers into a recess formed by etching away gold from an insulated goldwire.

The active polymer layers, including a sensing layer, a glucoseflux-limiting layer, a biocompatible layer, and optionally aperoxidase-based interferant eliminating layer, were protected withinthe recess against mechanical damage. (The peroxidase-based interferanteliminating layer is not required when a lower redox potential polymeris used, as described above.) The recess and its polymer layers alsoreduced the transport of glucose to the wire electrode contactingsensing layer.

By limiting the glucose flux, the desired linear response range,spanning the clinically relevant glucose concentration range wasobtained. The inventive biosensors are able to accurately measure, forexample, approximately 2-30 mμ glucose and approximately 0.5-10 mμlactate, in vivo. The sensor has no leachable components, and its fourcrosslinked polymer layers contain only about 5 μg of immobilizedmaterial, and only a few nanograms of polymer-bound osmium. Preoxidationof the interferants in one of the four layers makes possible one-pointin vivo calibration of the sensor.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic drawing of an electrode of the present invention.

FIG. 2 is a graphical representation of data generated comparing currentdensity of glucose electrooxidation on electrodes made with PVI₅-Os(open triangles) with those made with PVI₃-Os (filled triangles).

FIG. 3 is a graphical representation of data generated comparingdependency of current generated on the depth of the recess.

FIG. 4 is a graphical representation of data generated comparingdependency of the ratio of the current generated and the charge requiredto electoreduce or oxidize the polymer redox centers in the sensinglayer on the thickness of the sensing layer.

FIG. 5 is a graphical representation of data generated comparingvariation of current generated by electrodes having sensing layers ofdiffering thickness and diffusion limiting layers of differentcompositions and thickness. Solid circles: 7.5 μm thick sensing layer ofPVI₅-Os (52%), rGOX (35%), PEGDGE (13%), coated with 4 μm PAL/PAZ (1:1ratio). Open circles: 5.0 sensing layer. Solid triangles: 12.5 μmsensing layer and 7 μm PAL/PAZ (1:2 ratio). Open triangles: 7.5 μmsensing layer and 4.5 μm PAL/PAZ (1:2 ratio).

FIG. 6 is a graphical representation of data generated comparingdependency of current generated on the presence of ascorbate, in theabsence and presence of lactate and glucose. The concentrations ofascorbate (A), lactate (L) and glucose (G) are shown. Ascorbate is anelectrooxidizable interferant. Upon addition of lactate itselectrooxidation current is suppressed while that of glucose is notsuppressed.

FIG. 7 is a graphical representation of data showing current density andcorresponding subcutaneous glucose concentration measured with thesubcutaneously implanted electrodes of the present invention in a ratanimal model. Large solid circles show blood glucose concentrationsmeasured on withdrawn blood samples using a YSI analyzer.

FIG. 8 is a Clarke-type clinical grid analyzing the clinical relevanceof the blood glucose measurements of FIG. 7.

FIG. 9 is a Clarke-type clinical grid of all possible correlationsobtained when each of the 24 glucose analyses of FIG. 7 were used forsingle point calibration of either implanted electrode.

FIG. 10 is a Clarke-type clinical grid testing improvement of the singlepoint calibration through redundant electrodes, the readings of whichwere within the standard deviation calculated for all differencesbetween simultaneous readings by a pair of implanted electrodes.

DETAILED DESCRIPTION OF THE INVENTION

The present invention includes an insulated, non-corroding conductingmetal (e.g., gold, platinum, palladium) or carbon wire-based small(e.g., 290 μm) O.D. subcutaneous glucose sensor, allowing one-pointcalibration in vivo. As shown in FIG. 1, its construction involvescoating a small (e.g., 250 μm) diameter non-corroding metal or carbonwire 2 with an electrically insulating material 4, e.g., a polyimide,and, layering in a recess 6 formed by etching or removing a portion ofthe metal or carbon, the following active polymeric layers: animmobilized, “wired,” glucose oxidase layer 8; an electricallyinsulating and glucose diffusion limiting layer 10 formed, for example,by crosslinking a polyallylamine (PAL) with a polyaziridine (PAZ);optionally, an interference eliminating layer 12, e.g., of crosslinkedhorseradish-peroxidase and lactate oxidase; and a biocompatible film 14e.g., of poly(ethylene oxide) (PEO) derivatized to allow itsphoto-crosslinking. The outside diameter a of the wire 2 is preferablyabout 0.25 mm or less, and the outside diameter b of the insulated wireis preferably about 0.3 mm or less. The recess 6 in the insulatedelectrode extends from the tip 16 of the electrode which is open to thesurrounding environment, to the top 18 of the wire 2 in the insulatingsheath, generally for a length c of less than about 0.150 mm, andpreferably about 0.125 mm.

The electrodes have no leachable components. The total amount ofpolymers and enzymes is preferably about 5 μg. The glucose responsethrough the physiologically relevant 2-20 mM concentration range isclose to linear. The electrodes do not respond to ascorbate, urate oracetaminophenol for at least about 36 hours. Their 10-90% response timeis about 90 seconds at 2 mM glucose and about 30 seconds at 20 mMglucose. Their sensitivity, after about 30 minutes equilibration, isstable for about 72 hours at 37° C. in 10 mM glucose, the currentdeviating from the average by less than .+−0.5%. The electrodes havesubstantially no signal output, e.g., current, charge, or potential,when the concentration of the analyte to be measured is zero.

Two electrodes implanted subcutaneously in a rat tracked blood glucoselevels, and their absolute, uncorrected current output was proportionalto the blood glucose concentration. Analysis of the correlation betweenthe blood glucose levels in the tail vein and the current output of thesensors in the subcutaneous regions of the thorax and between thescapulae of the same rat showed that even when the probed sites andorgans differed in the extreme, one point in vivo calibration was valid.The analysis also showed the value of implanting redundant sensors. Hadclinical decisions been made based on individual sensor readings,calibrated at one point, 94% would have been clinically correct. Byusing redundant sensors and accepting only those pairs of readings thatwere within one standard deviation, the percentage of the clinicallycorrect decisions was increased to 99%.

It is understood that one of skill in the art may substitute variouscomponents of the biosensor described above with known materials toobtain an modified biosensor using the principles outlined herein. Forexample, the following substitutions are contemplated:

Base electrode: The base electrode of the inventive sensor may be formedof a non-corroding metal or carbon wire, for example vitreous carbon,graphite, platinum, palladium, or gold. Gold is preferred, and is usedin the following illustrative examples of the invention.

Insulator: The conductive metal or carbon wire is coated with anelectrically insulating material, which also forms a wall about therecess which houses the active polymeric components. The insulatingmaterial may be, for example, polyurethane, teflon (fluorinatedpolymers), polyethyleneterephthalate (PET, Dacron) or polyimide. Theinsulating material is preferably a biocompatible polymer containingless than about 5% water when in equilibrium with physiological bodyfluids, e.g., subcutaneous tissue.

Recess: In general, the recess at the tip of the electrode isapproximately 20 to 150 μm in length c, and preferably is approximately50 to 125 μm.

Etching method: The method for etching metal from the tip of theelectrode described herein may utilize chloride, bromide or iodide inthe bath in lieu of cyanide as described. Bromide is preferred, becauseit is less toxic and, like Au(CN)₂ ⁻, AuBr₄ ⁻ is a water soluble anion.Thus, in aqueous HBR, the metal, e.g., gold, an be etched by applying asufficiently oxidizing potential where gold is electrolyticallydissolved:

Au+4HBr

HAuBr₄+(3/2)H₂

Wired Enzyme Layer: In the sensing enzyme-containing layer, glucoseoxidase may be substituted with other redox enzymes to measure otherrelevant clinical compounds. For example, lactate oxidase may be usedfor the in vivo detection of lactate, important in determining if anorgan is receiving sufficient oxygen through the blood.

Useful redox polymers and methods for producing the sensing layer aredescribed, for example, in U.S. Pat. Nos. 5,264,104; 5,356,786;5,262,035, and 5,320,725. Additional redox polymers include, forexample, poly(1-vinyl imidazole); poly(4-vinyl pyridine); or copolymersof 1-vinyl imidazole such as poly(acrylamide co-1-vinyl imidazole) wherethe imidazole or pyridine complexes with [Os(bpy)₂Cl]^(+/2+);[Os(4,4′-dimethyl bipyridine)₂Cl]^(+/2+); [Os(4,4′-dimethylphenanthroline)₂Cl—]^(+/2+); [Os (4,4′-dimethoxyphenanthroline)₂Cl]^(+/2+); and [Os (4,4′-dimethoxybipyridine)₂Cl]^(+/2+); to imidazole rings. The imidazole ring compoundsare preferred because their complexes have more reducing redoxpotentials, i.e., closer to that of the SCE potential. At these morereducing potentials, the rate of electrooxidation of interferants andthe current generated thereby.

Barrier Layer: The polymeric barrier layer is electrically insulatingand limits diffusion of glucose through to the sensing layer. It may beformed, for example, by crosslinking a polyallylamine (PAL) with apolyaziridine (PAZ). Alternatively, PAL may be replaced wholly or inpart with a zwitterionic polymer obtained by quaternizingpoly(vinylpyridine) with bromoacetate and dialyzing against 0.15M NaClor by a polyanion such as a polysulfonic acid.

The barrier layer may contain a polyanionic polymer, in which the rateof permeation of anionic interferants such as ascorbate and urate isslowed. This layer may also contain a polycation that enhances theretention of the polyanion by electrostatic bonds and improves wettingby the biocompatible layer.

Interference Eliminating Layer: As described above, this layer isoptional, in that it is not required when a redox polymer having a morereducing potential is used, such as PVI₁₅-dmeOs (Ohara et al.,Analytical Chemistry, 1994, 64:2451-2457). At operating potentials ofapproximately −0.10 to +0.25 for the glucose biosensor, the rate ofelectrooxidation of interferants such as ascorbate, urate andacetaminophen is very slow relative to that of glucose through itsphysiological concentration range.

When a separate interferant eliminating layer is used, it preferablycontains a peroxidase enzyme which may or may-not be preactivated. Suchinterferant eliminating layers are disclosed, for example, in U.S. Pat.No. 5,356,786, which discloses the structure and function of interferanteliminating biosensors. The glucose biosensor preferably containslactate oxidase (LOX) in combination with peroxidase in the interferanteliminating layer. However, for biosensors used to detect lactate,glucose oxidase would be used with peroxidase. In a similar manner, theenzyme composition of the interferant eliminating layer may be alteredfor a specified function.

Biocompatible Layer: In general, the biocompatible layer is comprised ofhydrogels, e.g., polymeric compositions which contain more than about20% by weight of water when in equilibrium with a physiologicalenvironment such as living tissue or blood. An example is crosslinkedpoly(ethylene oxide), e.g., poly(ethylene oxide) tetraacrylate. Thepolymeric compositions must be non-toxic and compatible with livingSystems.

Method for making multi-layered recessed biosensors: Insulatednon-corroding metal or carbon wires that have been etched as describedabove to contain a recess at the tip, are placed in a block that servesas an X-Y positioner. The wires vertically traverse the block and areheld in place, e.g., by pressure. The blocks with the wires can beformed of elements, each element having multiple half-cylinder groovesrunning vertically. The wires are placed in these grooves and theelements are assembled into the block using screws. For example, theblock may be formed of aluminum having equally spaced holes, (900 for a30×30 array of wires), each hole to contain one wire. The block ispositioned under a fixed micronozzle that ejects a fluid in to therecess of the insulated wire.

To reduce the requirement of precision in the positioning of the blockand the micronozzle, the nozzle is electrically charged, with the wirehaving an opposite charge, or the wire being grounded or at least havinga potential such that there is a potential difference between the nozzleand the wire. Because the nozzle is charged, the microdroplets it ejectsare also charged with the same type of charge (positive or negative) asthe nozzle. The higher the potential on the nozzle (e.g., versus groundpotential), the higher the charge on the ejected microdroplets. If thetip of the wire to be coated is at ground potential or has a charge ofthe opposite type, the charged microdroplets are guided into the recessto deposit on the electrode, even if the jet of microdroplets is notvertical, i.e., even if the micronozzle is not precisely aligned abovethe wire's tip.

Furthermore, the higher the electrical potential on the nozzle (relativeto ground) the greater the charge on the ejected microdroplet. When thecharge is high enough, the droplet breaks up into two or more smallerdroplets because of electrostatic repulsion of charges on the droplet.Thus, the very small droplets all “drift” (drift meaning transportassisted by an electrical field) to the recessed electrode surface andare collected on it, even if they did not originate in a nozzleprecisely aligned with the electrode.

This coating method is useful in making any small biosensor, not onlythose in recessed zones.

Clinical Use of the Recessed Biosensors:

The recessed biosensors of the present invention have sufficientsensitivity and stability to be used as very small, subcutaneousbiosensors for the measurement of clinically relevant compounds such asglucose and lactate. The electrodes accurately measure glucose in therange of about 2-30 μM and lactate in the range of about 0.5-10 mM. Onefunction of the implanted biosensor is to sound an alarm when, forexample, a patient's glucose concentration is too low or too high. Whenpairs of implanted electrodes are used, there are three situations inwhich an alarm is triggered: low glucose concentration, high glucoseconcentration; sensor malfunction as determined by a discrepancy betweenpaired readings of the two sensors. A discrepancy sufficient to triggerthe alarm may be, for example more than two or three times the standarddeviation persisting for a defined period, e.g., not less than tenminutes. Such a system may be useful in sleeping patients, and also inemergency and intensive care hospital rooms, where vital functions arecontinuously monitored.

Another function of the inventive biosensors in to assist diabetics inmaintaining their blood glucose levels near normal. Many diabetics nowmaintain higher than normal blood glucose levels because of danger ofcoma and death in severe hypoglycemia. However, maintaining bloodglucose levels substantially, e.g., approximately 40% or more abovenormal leads to retinopathy and blindness as well as to kidney failure.Use of the subcutaneous biosensors to frequently, if not continuously,monitor glucose concentrations is desirable so that glucoseconcentrations can be maintained closer to an optimum level.

The subcutaneous biosensors can be used to measure the rate of rise anddecline of glucose concentrations after a meal or the administration ofglucose (e.g., a glucose tolerance test). The sensors are also useful infeedback loops for automatic or manually controlled maintenance ofglucose concentrations within a defined range. For example, when used inconjunction with an insulin pump, a specified amount of insulin isdelivered from the pump if the sensor glucose reading is above a setvalue.

In all of these applications, the ability to promptly confirm that theimplanted sensor reading is accurate is essential. Prompt confirmationand rapid recalibration are possible only when one-point calibration isvalid. Generally, even if a sensor's response is linear through therelevant concentration range, calibration requires at least two blood orfluid samples, withdrawn from the patient at times when the glucoseconcentration differs. It usually takes several hours for the glucoseconcentration to change sufficiently to validate proper functioning bytwo-point calibration. The ability to confirm and recalibrate using onlyone point is thus a highly desirable feature of the present invention.

Redundant sensors (e.g., at least two) are preferred in the clinicalapplication of the subcutaneous biosensors. Such redundancy permitssignaling of failure of any one sensor by recognition of an increase inthe discrepancy between the readings of the sensors at one time point,e.g., more than two standard deviations apart. The redundant sensors maybe implanted near each other or at remote sites.

It is preferred that the biosensors be implanted in subcutaneous tissueso as to make the sensor relatively unobtrusive, and at a site wherethey would not be easily dislodged, e.g., with turning or movement. Itis also preferred, when readings are not corrected for temperature(which they generally are) that the sensors be implanted where they arelikely to be at body temperature, e.g., near 37° C., and preferablycovered by clothing. Convenient sites include the abdomen, inner thigh,arm.

Although we describe here continuous current measurement for assayingglucose, the electrical measurement by which the glucose concentrationis monitored can be continuous or pulsed. It can be a currentmeasurement, a potential measurement or a measurement of charge. It canbe a steady state measurement, where a current or potential that doesnot substantially change during the measurement is monitored, or it canbe a dynamic measurement, e.g., one in which the rate of current orpotential change in a given time period is monitored. These measurementsrequire at least one electrode in addition to the sensing electrode.This second electrode can be placed on the skin or can be implanted,e.g., subcutaneously. When a current is measured it is useful to have apotentiostat in the circuit connecting the implanted sensing electrodeand the second electrode, that can be a reference electrode, such as anAg/AgCl electrode. When a current is measured the reference electrodemay serve also as the counter electrode. The counter electrode can alsobe a separate, third electrode, such as a platinum, carbon, palladium orgold electrode.

In addition to implanting the sending electrode in the body, fluid fromthe body, particularly fluid from the subcutaneous region, can be routedto an external sensor. It is preferred in this case to implant in thesubcutaneous region a microfiltration giver and pull fluid to anevacuated container, the fluid traversing a cell containing the sensingelectrode. Preferably this cell also contains a second electrode, e.g.,a reference electrode which may serve also as a counter electrode.Alternatively, the reference and counter electrodes may be separateelectrodes. In coulometric measurements only two electrodes, the sensingelectrode and the counter electrode are required. The flow of body fluidmay be pulsed or continuous. Other than an implanted microfiltrationfiber, also a microdialysis fiber may be used, preferably in conjunctionwith a pump.

Increased Stability of the Biosensors:

To increase the stability and useful life of the inventive biosensors,it is advantageous to use intrinsically more stable enzymes and redoxpolymers. However, even if the enzyme and redox polymer degrade in theglucose electrooxidation process by which the signal (current) isgenerated, it is possible to greatly extend the useful life of theimplanted electrodes and reduce the frequency of their requiredrecalibration after implantation.

A simple measure by which the life of the implanted electrodes can beextended and the frequency of their required recalibration reducedinvolves turning the electrodes “on” by applying a bias, i.e., apotential, only during the period of measurement, then turning thebiasing potential off or reducing it, so that a lesser current willflow. It is generally sufficient to perform only one measurement everyfive or even ten minutes, or longer, because glucose concentrations donot change abruptly.

Another measure is to lower the glucose flux to the sensing layer muchas possible, consistent with maintaining adequate sensitivity anddetectivity. Reduction of the glucose flux to the sensing layer reducesthe current. Therefore, even though this stabilizes the electrodes,i.e., slows the loss in sensitivity, the flux dependent current must notbe excessively reduced. Usually a current of 3-5 nA at 2 mM glucoseconcentration is adequate. When the glucose flux is lowered by using oneor more glucose-flux reducing polymer slayers, such as the PAL/PAZlayer, the lifetime of the sensor is increased.

EXAMPLES Example 1 Electrode Preparation

Electrodes were made of a polyamide-insulated 250 μm diameter gold wire,having an outer diameter (O.D.) of 290 μm (California Fine Wire Co.,Grover City, Calif.). Heat shrinkable tubing (RNF 100 3/64″ BK and 1/16″BK, Thermofit®, Raychem, Menlo Park, Calif.) and a two component silverepoxy (Epo-tek H₂OE; Epoxy Tech, Inc., Billerica, Mass.) were used forelectrode preparation.

The glucose sensing layer was made by crosslinking a geneticallyengineered glucose oxidase (rGOX) (35% purity, Chiron Corp., Emeryville,Calif.) with a polymer derived of poly(vinylimidazole) (PVI), made bycomplexing part of the imidazoles to [Os(bpy)₂Cl]^(+/2+). The resultingredox polymer, termed PVI-Os, was synthesized according to a previouslypublished protocol. (Ohara et al., 1993, Anal. Chem., 65:24).Poly(ethylene glycol)diglycidyl ether 400 (PEDGE; Polysciences,Warrington, Pa.) was used as the crosslinker.

The barrier layer between the sensing and interference-eliminatinglayers was made of polyallylamine (PAL; Polysciences) crosslinked with apolyfunctional aziridine (PAZ) (XAMA-7; Virginia Chemicals, Portsmouth,Va.).

The interference-eliminating layer was prepared by co-immobilizinghorseradish peroxidase (HRP) type VI (Cat. No. P-8375, 310 U/mg, denotedherein as HRP-VI, Sigma, St. Louis, Mo.) and HRP for immunological assay(No. 814407, min 1000 U/mg, denoted HRP-BM, Boehringer-Mannheim,Indianapolis, Ind.) with lactate oxidase from Pediococcus sp. (Cat. No.1361, 40 U/mg denoted LOX, Genzyme, Cambridge, Mass.) and a recombinantmicrobial source (Cat. No. 1381 denoted rLOX, Genzyme).Co-immobilization was performed using sodium periodate (Cat. No. S-1147,Sigma) according to the methods described in Maidan and Heller, 1992,Anal. Chem. 64:2889-2896.

The biocompatible layer was made of 10% aqueous poly(ethylene oxide)tetraacrylate (PEO-TA). To form the photocrosslinkable polymer, PEO wasacrylated by reaction with acryloyl chloride. The 18,500 g/mol PEO(Polysciences) is a tetrahydroxylated compound by virtue of two hydroxylgroups on a bisphenol A bisepoxide that linked two .alpha.,.omega.-hydroxy-terminated 9,000 g/mol PEO units. Acryloyl chloride(Aldrich, Milwaukee, Wis.) in a 2 to 5 molar excess was used to acrylatethe polymer (10% w/v PEO in benzene). Triethylamine (Mallinkrodt, Paris,Ky.) was used as a proton acceptor equimolar with the acryloyl chloride.

Other chemicals used were bovine serum albumin (BSA) fraction V (Cat.No. A-2153), BSA, ascorbic acid, uric acid, 4-acetaminophenol,L(+)=lactic acid, and hydrogen peroxide 30%, all from Sigma. Allchemicals were used as received. Solutions (if not otherwise specified)were made with distilled, deionized water. Glucose monitoring wasperformed in buffer, in bovine serum (Sigma, Cat. No. S-6648) containingantibiotic-antimycotic solution (Sigma, Cat. No. A-8909) at 37° C. andin rats.

Instrumentation

In making the recessed gold electrodes, a potentiostat/galvanostat (PARModel 173, Princeton Applied Research, Princeton, N.J.) operated in agalvanostatic mode, and a sonicator (Fisher scientific, Pittsburgh, Pa.)were used. Cyclic voltammograms were recorded with a potentiostat (PARModel 273A) and a conventional electrochemical cell having a Pt wirecounter and a SCE reference electrode and were evaluated with PAR 270software. Glucose signals were monitored with a bipotentiostat (BiometraEP 30) and a two channel strip-chart recorder. The recessed electrodeswere coated under a microscope (Bausch & Lomb) using a micromanipulator(Narishige, Seacliff, N.Y.). The micropipettes were pulled with amicropipette puller (Narishige). Temperature was controlled with anisothermal circulator (Fisher Scientific).

Electrode Preparation:

Five cm lengths of polyamide insulated gold wire were cut with a sharprazor blade. Electrical contact was made at one end with silver epoxy toan insulated stainless steel wire and the junction was covered withinsulating heat shrinkable tubing. The recess forming electrochemicaletching process was carried out in 10 ml of 3M potassium cyanide, withthe gold wire as the working electrode and a platinum or gold wire asthe counter electrode. The wires were placed in contact with the bottomof the beaker, all electrodes being equidistant from the counterelectrode. The beaker was sonicated during the etching procedure. Theends of the gold wires were bent upwards, so that agitation by thesonicator caused the oxygen bubbles formed during the etching process torise and escape. The electrodes were then thoroughly washed and immersedin water for 30 minutes.

A recess 6, i.e., channel, in a polyamide insulated gold wire 2 isformed by electrochemical etching of the gold under galvanostaticcontrol. By controlling the charge, the total amount of goldelectrooxidized and dissolved as Au(CN)₂ is defined.

When the conditions were set so that the CN— transport into the channeland the Au(CN)₂— transport out of it are not rate limiting, (e.g.,sonicated bath and high concentration of potassium cyanide, at leastapproximately 0.2M, and preferably 3M), a flat gold wire surface isproduced at the bottom of channels with aspect ratios of 0.5 to 2.0.Thus, when the CN— concentration is high enough and the wires areultrasonically vibrated, the tips of gold wires are flat. Passage of 1.5coulombs per electrode at 8 mA current produced approximately 125 μmdeep cavities or channels. At theoretical efficiency for one-electronoxidation, 3.08 mg of gold would have been etched. The amount of goldactually etched was only 0.076 mg, showing significant CN— or wateroxidation.

Nevertheless, the process is reproducible, accurate and fast with 20electrodes being processed in each batch in less than five minutes. Therecess-forming procedure was highly reproducible, with a deviation of+/−10 μm found (using an objective micrometer) for a batch of 30recessed electrodes. Before coating, the electrodes were examined undera microscope for flatness of the gold surface and correct depth.

FIG. 1 shows a schematic side view in cross-section of an electrode ofthe present invention, showing the gold wire 2, insulating coating 4,and recess or channel 6. The recessed gold surfaces were coated byfilling of the cavities or channels 6 with aqueous solutions containingthe crosslinkable components of the different layers, and theircrosslinkers. The solutions were introduced under a microscope with amicropipette (connected to a microsyringe by polyethylene tubing andshrink tubing), using a micromanipulator. After application of each ofthe individual layers, the electrodes were cured overnight at roomtemperature, in air.

Electrode Structure:

The electrodes were prepared by sequentially depositing four layerswithin the recess or channel 6. The layers were: the sensing layer 8,the insulating layer 10, the interference-eliminating layer 12 and thebiocompatible layer 14. The sensing layer, containing “wired” redoxenzyme is positioned adjacent to and in contact with the gold wire 2.The insulating layer 10 is positioned between the sensing layer 8 andthe peroxidase-based interferant-eliminating layer 12. The biocompatiblelayer 14 fills the remaining space in the recess 6 and is in contactwith the environment outside the electrode. The thin polymer layers arewell protected by containment within the polyamide sleeve 4.

The sensing layer 8 was made by “wiring” rGOX to the gold electrodethrough a redox hydrogel to which the enzyme was covalently bound. Theelectrodes were prepared as follows: 10 mg/ml solutions were made from

1. the PVI-Os redox polymer in water,

2. the crosslinker, PEGDGE, in water, and

3. the enzyme, rGOX, in a 10 mM HEPES solution adjusted to pH 8.15.

A redox hydrogel was formed by mixing the three solutions so that thefinal composition (by weight) was 52% redox polymer, 35% enzyme and 13%crosslinker.

The insulating layer 10 prevented electrical contact between the redoxhydrogel and the interference eliminating enzymes (HRP and LOX). PAL:PAZwas used as the insulating material. The film was deposited from asolution obtained by mixing in volume ratio of 1/1, 1/2 or 1/3, a PALsolution (4.5 mg in 100 mM HEPES buffer at pH 7.0) and a freshlyprepared PAZ solution (30 mg/ml). The PAZ solution was used within 15minutes of preparation.

The interference-eliminating layer 12 was prepared according to apreviously published protocol, Maidan and Heller, 1992, Anal. Chem.,64:2889-2896. 50 μl of a 12 mg/ml freshly prepared sodium periodatesolution was added to 100 μl of a solution containing 20 mg/ml HRP(HRP-VI or HRP-BM) and 100 mg/ml LOX (LOX or rLOX) in 0.1 M sodiumbicarbonate and the mixture was incubated in the dark for two hours.Alternatively, the oxidation of HRP could be carried out prior to addingLOX and cross linking.

The biocompatible layer 14 films were photocrosslinked by exposure to UVlight (UVP, Inc., San Gabriel, Calif.; Blak-Ray; spectral peak at 360 nMUV irradiance at the sample 200 mW/cm²) for one minute. The initiatorused was 2,2-dimethoxy-2-phenylacetophenone (Aldrich). A solution of 300mg/ml of the initiator in 1-vinyl-2-pyrrolidinone (Aldrich) was added tothe prepolymer mixtures. Approximately 30 μl of the initiator solutionwas added per ml of 10% w/w aqueous solution of the tetraacrylated PEO.The prepolymers were crosslinked in situ inside the recess of theelectrode. The films were prepared by filling the recess with theprepolymer solution twice and exposing the electrode to the UV lightsource after each time the cavity was filled.

In Vitro Testing of Electrodes:

In vitro experiments were carried out in batch fashion at 25° and 37°C., using a conventional three electrode electrochemical cell with theenzyme-modified gold wire as the working electrode, a platinum wire asthe counter electrode and a saturated calomel reference electrode (SCE).The electrolyte was a 20 mM phosphate buffered-saline solutioncontaining 0.15 M NaCl at pH 7.15. Experiments in serum were performedat 37° C., adding 100 μL antibiotic-antimycotic solution to 10 ml serum.Phosphate buffered-saline and serum were agitated during theexperiments. The working potential was +0.3 V versus SCE for experimentswith the PVI-Os polymers.

Structure and Performance: The depth c of the channel 6 and thethickness of the polymer layers in it controls the mass transport, i.e.,flux of glucose, to the sensing layer. By controlling these parameters,the apparent Michaelis constant (K_(m)) is adjusted to about 20-30 mMglucose. The polyimide wall 4 of the channel 6 also protects the fourpolymer and polymer/enzyme layers 8, 10, 12, 14 against mechanicaldamage and reduces the hazard of their loss in the body. Because theglucose electrooxidation current is limited by glucose mass transportthrough the recess 16 and its polymer films 8, 10, 12, 14, rather thanby mass transport to the tissue-exposed tip 16, the current ispractically insensitive to motion. Evidently, the electrooxidation rateof glucose in the recessed sensing layer 8 is slower than the rate ofglucose diffusion to the channel's outer fluid contacting interface.

PVI₅-Os is preferred as the “wire” of the sensing layer when aninterference eliminating layer of HRP and LOX is used, but not in theabsence of this layer, i.e., when redox polymers with more reducingredox potential are preferred. The subscript (5) is used to indicatethat, on the average, every fifth vinylimidazole mer carries anelectron-relaying osmium center. Use of electrodes formed with PVI₅-Osand PVI₃-Os (every third 1-vinylimidazole mer carrying an osmium center)are compared in FIG. 2, and show higher current density of glucoseelectrooxidation on electrodes made with PVI₅-Os (open triangle) than onthose made with PVI₃-Os (filled triangles).

Depth of the recess and the sensing layer: Channels of 125, 250, and 500μm depth, were investigated to assess the dependence of the current onthe depth of the recess (FIG. 3), with the total amount of PVI₅-Os andrGOX being kept constant. Much of the loss in current in the deepercavities resulted not from reduced glucose mass transport, but fromadsorptive retention of part of the enzyme and polymer on the polyamidewall when microdrops of the component solutions were introduced into therecess in the process of making the electrodes. Through repeated rinsingwith water, some of the adsorbed polymer and enzyme on the walls werewashed onto the electrode surface, increasing the current. The highestcurrents were seen after five washings. When the thickness of thesensing layer was increased through increasing the number of coatings(FIG. 4) the ratio of current to charge required to electroreduce orelectrooxidize the redox polymer in the sensing layer reached a maximum,then dropped. For the preferred 125 μm recess, 10 coatings, producing anapproximately 13 Am thick wired-rGOX sensing layer, yielded sensors thathad the desired characteristics for in vivo use.

The insulating layer: This layer electrically insulates the redoxenzymes of the interference eliminating layer (HRP and LOX) from the“wired” rGOX layer and limits the glucose flux to the sensing layer,thereby extending the useful life of the electrode. PAL crosslinked withPAZ, forming a polycationic network at pH 7.09 is preferred. The bestresults, i.e., best stability of current outputs, were obtained using1:2 PAL:PAZ (FIG. 5), with three coatings applied to form anapproximately 7 μm thick crosslinked film.

The interference eliminating layer: Interferants, particularlyascorbate, urate, and acetaminophenol, are oxidized in the third layer,containing LOX and HRP. In this layer, lactate, the typicalconcentration of which in blood is 1 mM, reacts with O₂ to form H₂O₂ andpyruvate. H₂O₂, in the presence of HRP, oxidizes ascorbate, urate, andacetaminophenol, being reduced to water. The preferred coimmobilizationprocess involved two separate steps: periodate oxidation ofoligosaccharide functions of HRP to aldehydes, followed by mixing withLOX and formation of multiple Schiff bases between HRP-aldehydes and LOXamines (e.g. lysines) and between HRP aldehydes and amines. Thethickness of the interference eliminating layer is approximately 85 μmand is made by applying successive coatings, e.g., about six coatings.FIG. 6 shows that electrooxidizable interferants were eliminated in thepresence of lactate at physiological levels. LOX slowly lost itsactivity in the crosslinked HRP-LOX layer. This led to degradation ofthe ability of the layer to eliminate interferants. After 36 hours ofoperation at 37° C., a measurable current increment was noted whenenough ascorbate was added to produce a 0.1 mM concentration.

The biocompatible layer: A preferred biocompatible layer consists, forexample, of photocrosslinked tetraacrylated 18,500 Da poly(ethyleneoxide) (Pathak et al., 1993, J. Am. Chem. Soc., 114:8311-8312). Thethickness of this layer, made by sequential photocrosslinking of twocoatings, is about 20 μm. One minute UV exposure required for thephotocrosslinking process reduced the sensitivity by 16+/−2%.

Example 2 In Vivo Use of Sensor

The objective of this experiment was to establish the validity of aone-point in vivo calibration. Two sensors were simultaneously implantedsubcutaneously in a rat, one on the thorax, the second between thescapulae. To make the difference between the blood sampled and thesubcutaneous fluid proved with the sensors as extreme as possible, i.e.,to probe whether the one-point calibration holds even if the organssampled are different and the sampling sites are remote, blood waswithdrawn from the tail vein. Blood glucose levels were periodicallymeasured in withdrawn samples, while the absolute uncorrected sensorcurrent output was continuously monitored.

In vivo experiments (6-10 hours) were carried out in 300 g maleSprague-Dawley rats. The rats were fasted overnight and prior to theexperiment were anaesthetized with an intraperitoneal (i.p.) injectionof sodium pentobarbital (65 mg/kg rat wt). An i.p. injection of atropinesulfate (166 mg/kg rat wt) was then administered to suppress respiratorydepression. Once the rat was anaesthetized, a portion of the rat'sabdomen was shaved, coated with a conductive gel, and an Ag/AgCl surfaceskin reference electrode was attached. This electrode served also as thecounter electrode. Sensors were then implanted subcutaneously using a 22gauge Per-Q-Cath Introducer (Gesco International, San Antonio, Tex.) onthe rat's thorax, or subcutaneously in the intrascepular area through asmall surgical incision. The sensors were taped to the skin to avoidsensor movement. The sensors, along with the reference electrode, wereconnected to an in-house built bipotentiostat. The operating potentialof the sensors was 0.3 V vs. Ag/AgCl, with the Ag/AgCl electrode servingas both the reference counter electrode. Sensor readings were collectedusing a data logger (Rustrak Ranger, East Greenwich, R.I.) and at theend of the experiment were transferred to a computer. During theexperiment, the rat's body temperature was maintained at 37° C. by ahomeostatic blanket. The sensors were allowed to reach a basal signallevel for at least one hour before blood sampling was started. Bloodsamples were obtained from the tail vein and all blood samples wereanalyzed using a glucose analyzer (YSI, Inc., Yellow Springs, Ohio;Model 23A).

Approximately thirty minutes after the start of blood sampling, an i.p.glucose infusion was started using a syringe pump (Harvard Apparatus,South Natick, Mass.) at a rate of 120 mg glucose/min kg rat wt. Theglucose infusion was maintained for approximately one hour.

As seen in FIG. 7, at 410 min the current dropped precipitously. Such adrop was observed in other measurements with subcutaneously implantedelectrodes between 400 and 600 min, but was never observed in electrodesoperated in buffer at 37° C. When the failed electrodes were withdrawnand retested in buffer, most of their original sensitivity was found tobe intact. The cause for this apparent deactivation was failure of thecounter/reference Ag/AgCl electrode on the rat's skin to make goodelectrolytic contact, and was not due to any failure of the implantedsensor. Using an arbitrarily chosen point to calculate a calibrationcurve for each electrode, i.e., one blood glucose level determinationand one current measurement to establish the scales, all the data fromFIG. 7 were plotted in a Clarke-type, (Clarke et al., 1987, DiabetesCare, 5:622-627) clinical grid (FIG. 8), without further correction. Inthis analysis, points falling in region A of the grid are consideredclinically accurate, while those in region B are considered clinicallycorrect. Points falling in region C are not correct, but would not leadto improper treatment. Points in regions D and E are incorrect and iftreatment would rely on these, it would be improper.

All of the points, from both electrodes, were in regions A and B, with43 of the 48 points being in region A. The three points in region B near100 mg/dl glucose, for the electrode implanted between the scapulae,were the last three points of the experiment, at about 410 min.Notwithstanding the failure mode at 400-600 min because of poorelectrolytic contact of the counter/reference electrode with the skinand failure after 36 hours by deactivation of the lactate oxidase,resulting in loss of interference elimination, one-point calibration isshown here to be practical. After such calibration, the readings of thesubcutaneous sensors provide, without any correction, clinically usefulestimates of blood glucose levels.

FIG. 9 shows the distribution of all possible. correlations obtainedwhen each of the 24 glucose analyses was used for single pointcalibration of either implanted electrode. There are 2×24×24=1152 pointsin the distribution. Of these, 78% are in region A, 15% are in region B,1% in region C, 6% are in region D, and no points are in region E.

In FIG. 10, we tested for the improvement of the single pointcalibration through using redundant electrodes. First, the readings ofelectrode A were normalized with respect to those of electrode B bymultiplying each reading by the average output of electrode B divided bythe average output of electrode A. Next the standard deviation wascalculated for the differences between the 24 sets of readings ofimplanted electrode B and corrected readings of implanted electrode A.Then, all those sets of readings that differed by more than the standarddeviation were rejected. The number of sets was reduced thereby from 24to 11; 82% of the points were in region A, 17% in region B, 1% in regionD, and no points in regions C and E. The distribution demonstrates thatthe sensors can be calibrated through a single independent measurementof the glucose concentration in a withdrawn blood sample. They alsodemonstrate the improvement in clinical accuracy resulting from the useof redundant subcutaneous sensors. The selection of those data pointsthat differed by less than the standard deviation for the entire set ledto a sixfold reduction in the probability of clinically erring in adecision based on readings of the implanted sensors.

Stability and Other Characteristics:

In order to improve the stability, more thermostable recombinant GOX,(rGOX; Heller, 1992, J. Phys. Chem., 96:3579-3587) rather than GOX isused in the sensor and glucose transport is reduced to make the sensorcurrent diffusion, not enzyme turnover, limited. The glucose flux isattenuated by the three outer layers and the sensing layer itself.Because the sensing layer contains a large excess of glucose oxidase,its activity greatly exceeds that needed for electrooxidizing theattenuated glucose flux, and the sensor's stability is improved.

The stability can be tested by methods known, for example, tested in thepresence of 0.1 mM ascorbate in 10 mM glucose at 37° C. The currentoutput of a typical optimized electrode was about 35 nA and the apparentK_(m), derived from an Eadie-Hofstee plot, was about 20 mM (Table 1).The 10-90% response time was approximately one minute.

As expected, and as can be seen in FIG. 5, with thinner films theglucose mass transport was increased, i.e., the current was higher,while for thicker films the stability was improved. Because of the highsensitivity of thin sensing film (approximately 1 μm) electrodes (lessthan 10⁻²A cm⁻² M⁻¹), an order of magnitude decrease in sensitivitycould be traded for stability, while the currents remained high enoughto be easily measured.

As seen in FIG. 5, the sensitivity of the stabilized sensors does notchange by more than +5% for 72 hours of operation at 37° C. After asmall initial decrease in sensitivity, it increased to a maximum after40 hours and the final 72 hour sensitivity was almost identical with theinitial.

The characteristics of the electrodes of the present invention aresummarized in Table 1. Each entry represents an average value for fivetested electrodes. Baseline currents are typically less than 0.5 nA andthe noise less than 10 pA. The currents observed throughout thephysiological glucose concentration range (2-20 mM) exceed the noiseequivalent current by at least a factor of 100. The apparent K_(m) is 20mM, and the 10% to 90% response time is, for aged electrodes, about 90seconds at the lowest physiologically relevant glucose concentration (2mM) and 20 seconds at the highest (20 mM).

The baseline of nil at 0 mM glucose is stable for 36 hours in thepresence of 0.1 mM ascorbate. The stability observed and the existenceof a valid zero-point in the presence of interferants suggest that thesensor can be used in vivo for 72 hours and tested/recalibrated in vivothrough a single point calibration, i.e., by withdrawing only a singlesample of blood for independent analysis.

TABLE 1 SENSOR CHARACTERISTICS Current K_(m) ^(app) (mM) K_(m) ^(app)(mM) Variance i (nA) j (μA/cm²) EH LB t_(r) (s) (%) 33.9 69.1 18.5 33.430-90 5.0 where: I is the current measured at 37° C. and at 10 mMglucose concentration j is the current density measured at 37° C. at 10mM glucose concentration K_(M) ^(app) is the apparent Michaelis-Mentencoefficient determined from an electrochemical Eadie-Hoffstee (EH) orLineweaver-Burk (LB) plot t_(r) is the 10-90% risetime, 90 s for 2 mMand 30 s for 20 mH glucose concentration. Current Variance is themaximum deviation from the mean value, measured during the 72 hour test,conducted in 10 mM glucose in the presence of interferants. The currentwas continuously monitored at 37° C.

The foregoing examples are designed to illustrate certain aspects of thepresent invention. The examples are not intended to be comprehensive ofall features and all embodiments of the present invention, and shouldnot be construed as limiting the claims presented herein.

1. An electrochemical sensor for measuring an analyte in an animalcomprising: one or more non-corroding metal or carbon electrodes adaptedfor at least partial subcutaneous implantation in an animal; a sensinglayer comprising an enzyme disposed adjacent to each electrode; andwherein the electrochemical sensor has a glucose sensitivity selectedfrom the range of about an order of magnitude less than 61 pA/mg/dL toabout 300 pA/mg/dL.
 2. The electrochemical sensor of claim 1, whereinthe electrochemical sensor has a glucose sensitivity selected from therange of about an order of magnitude less than 61 pA/mg/dL to about anorder of magnitude less than 300 pA/mg/dL.
 3. The electrochemical sensorof claim 1, wherein the electrochemical sensor has a glucose sensitivityselected from the range of about an order of magnitude less than 97pA/mg/dL to about 167 pA/mg/dL.
 4. The electrochemical sensor of claim1, wherein the electrochemical sensor has a glucose sensitivity selectedfrom the range of about an order of magnitude less than 78 pA/mg/dL toabout 158 pA/mg/dL.
 5. The electrochemical sensor of claim 1, whereinthe electrochemical sensor has a glucose sensitivity of about an orderof magnitude less than 78 pA/mg/dL.
 6. The electrochemical sensor ofclaim 1, wherein the sensitivity does not change by more than about ±5%for 3 days of operation.
 7. The electrochemical sensor of claim 1,further comprising a biocompatible layer disposed adjacent to thesensing layer.
 8. The electrochemical sensor of claim 6, wherein thebiocompatible layer comprises a hydrogel.
 9. The electrochemical sensorof claim 8, wherein the hydrogel comprises more than about 20% by weightof water when in equilibrium with physiological body fluids.
 10. Theelectrochemical sensor of claim 8, wherein the hydrogel comprises acrosslinked poly(ethylene oxide).
 11. The electrochemical sensor ofclaim 6, wherein all portions of the sensing layer and the biocompatiblelayer allow diffusion of an analyte therethrough.
 12. Theelectrochemical sensor of claim 6, wherein the biocompatible layer ischemically bound to the sensing layer.
 13. The electrochemical sensor ofclaim 6, wherein the biocompatible layer is indirectly chemically boundto the sensing layer.
 14. The electrochemical sensor of claim 6, whereinthe sensor further comprises an interferant eliminating layer and thebiocompatible layer is chemically bound to the interferant eliminatinglayer.
 15. The electrochemical sensor of claim 6, wherein the sensorfurther comprises an analyte flux limiting layer and the biocompatiblelayer is chemically bound to the analyte flux limiting layer.
 16. Theelectrochemical sensor of claim 1, wherein the sensing layer comprises ahydrogel.
 17. The electrochemical sensor of claim 16, wherein thesensing layer comprises glucose oxidase.
 18. The electrochemical sensorof claim 1, wherein the outer dimension of the sensor is smaller thanabout 0.3 mm
 19. The electrochemical sensor of claim 1, wherein thenon-corroding metal or carbon electrode comprises a wire.
 20. Theelectrochemical sensor of claim 19, wherein the outer diameter of eachof the one or more non-corroding metal or carbon electrodes is smallerthan about 0.25 mm.
 21. The electrochemical sensor of claim 1, whereinthe electrochemical sensor further comprises an electrically insulatingmaterial coating at least a part of one of the one or more non-corrodingmetal or carbon electrodes.
 22. The electrochemical sensor of claim 21,wherein the electrically insulating material is selected from the groupconsisting of polyimide, polyester, polyurethane, and perfluorinatedpolymer.
 23. The electrochemical sensor of claim 21, wherein theelectrically insulating material disposed on said one or morenon-corroding metal or carbon electrodes defines a gap in the coatingexposing a non-insulated portion of the electrode.
 24. Theelectrochemical sensor of claim 23, wherein the sensing layer is atleast partially disposed on the exposed, non-insulated portion of theelectrode.
 25. An electrochemical sensor for measuring an analyte in ananimal, comprising: one or more analyte responsive electrodes, at leastone of said analyte-responsive electrodes adapted for subcutaneousimplantation in an animal, each of the analyte responsive electrodescomprising a non-corroding metal or carbon electrode, a sensing layercovering at least a portion of each non-corroding metal or carbonelectrode, comprising a redox enzyme and a redox compound, wherein theredox enzyme is non-leachable by fluids in the body of the animal at apH of between about 6.5 and about 7.8, and an alarm coupled to thenon-corroding metal or carbon electrode configured to activate if asignal generated at the electrode indicates at least one of impendinghypoglycemia and impending hyperglycemia.
 26. The electrochemical sensorof claim 25, wherein the one or more non-corroding metal or carbonelectrodes are flexible.
 27. The electrochemical sensor of claim 25,wherein the electrochemical sensor further comprises a biocompatiblelayer covering the sensing layer of each non-corroding metal or carbonelectrode.
 28. The electrochemical sensor of claim 25, wherein thesensor further comprises a biocompatible layer
 29. The electrochemicalsensor of claim 28, wherein the biocompatible layer comprises ahydrogel.
 30. The electrochemical sensor of claim 25, wherein thesensing layer comprises a hydrogel.
 31. The electrochemical sensor ofclaim 30, wherein the sensing layer comprises glucose oxidase.
 32. Theelectrochemical sensor of claim 25, wherein the outer dimension of thesensor is smaller than about 0.3 mm
 33. The electrochemical sensor ofclaim 25, wherein the non-corroding metal or carbon electrode comprisesa wire.
 34. The electrochemical sensor of claim 25, wherein the outerdiameter of each of the one or more non-corroding metal or carbonelectrodes is smaller than about 0.25 mm.
 35. The electrochemical sensorof claim 25, wherein the electrochemical sensor further comprises anelectrically insulating material coating at least a part of one of theone or more non-corroding metal or carbon electrodes.
 36. Theelectrochemical sensor of claim 35, wherein the electrically insulatingmaterial is selected from the group consisting of polyimide, polyester,polyurethane, and perfluorinated polymer.
 37. The electrochemical sensorof claim 35, wherein the electrically insulating material disposed onsaid one or more non-corroding metal or carbon electrodes defines a gapin the coating exposing a non-insulated portion of the electrode. 38.The electrochemical sensor of claim 37, wherein the sensing layer is atleast partially disposed on the exposed, non-insulated portion of theelectrode.
 39. A method of calibrating an electrochemical sensor atleast partially implanted in an animal, comprising the steps of:withdrawing a calibration sample from an animal during a time period,wherein the time period is selected such that the analyte concentrationremains substantially constant during the time period; assaying ananalyte concentration of the calibration sample; correlating the assayedanalyte concentration to a signal generated by one or more implantedworking electrodes of an electrochemical sensor to calibrate theelectrochemical sensor, each working electrode having ananalyte-responsive enzyme disposed thereon.
 40. The method of claim 39,wherein the sensor is capable of operation for 72 hours without furthercalibration.